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Alpha ketoglutarate

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Alpha-ketoglutarate is a chemical compound that serves as an important intermediate in the citric acid cycle, a metabolic pathway that plays a crucial role in cellular respiration. It is a key component in the production of energy within cells.

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Lab products found in correlation

Oxaloacetate, by Merck Group (5 mentions) Pyruvate, by Merck Group (4 mentions) Adenosine triphosphate (atp), by Merck Group (4 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions) Dimethyl alpha ketoglutarate, by Merck Group (3 mentions) Malate, by Merck Group (3 mentions) Succinate, by Merck Group (3 mentions) Tridecanoic acid, by Merck Group (3 mentions) Amitriptyline, by MP Biomedicals (3 mentions) Succinic acid, by MP Biomedicals (3 mentions) Pyruvic acid, by MP Biomedicals (3 mentions) Oxaloacetic acid, by Merck Group (3 mentions) Malic acid, by Merck Group (3 mentions) D9 progesterone, by CDN Isotopes (3 mentions) D7 glucose, by Cambridge Isotopes (3 mentions) D3 leucine, by Cambridge Isotopes (3 mentions) Phenylalanine d8, by Cambridge Isotopes (3 mentions) Tryptophan d5, by Cambridge Isotopes (3 mentions) D5 hippuric acid, by CDN Isotopes (3 mentions)

Close spelling variants of this product

Dimethyl-alpha-ketoglutarate (7 citations)

17 protocols using alpha ketoglutarate

1

Molecular Inhibitors for Cell Signaling

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Chemical inhibitors used included 1uM MG132 (S2619; Selleckchem), 1.3 uM Chir911, 1 uM Rapamycin (S1039, Selleckchem), 1uM Torin2 (S2817; Selleckchem), 1 ug/mL DON (D2141, Sigma-Aldrich), 1 ug/mL alpha keto-glutarate (K2000; Sigma-Aldrich), 0.1 mM actinomycin D (114666; Calbiochem), 100 ug/mL cyclohexamide (C7698; Sigma-Aldrich), 1uM 2DG (D6134; Sigma-Aldrich), and 1 or 5 uM JQ1 (A1910; Apexbio). Cytokines included recombinant murine IL-7 (5 ng/mL) (217-17; Peprotech), recombinant murine IL-2 (5 ng/mL) (212-12; Peprotech), and recombinant murine GMCSF for BMDC generation (1000 U/mL) (31503; Peprotech).
Verbist K.C., Guy C.S., Milasta S., Liedmann S., Kamiński M.M., Wang R, & Green D.R. (2016). Metabolic Maintenance of Cell Asymmetry following Division in Activated T Lymphocytes. Nature, 532(7599), 389-393.
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2

Apoptosis and Proliferation Assays

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The following materials were used: media—RPMI-1640, FBS, DMEM, and antibiotic–antimycotic solution (Thermofisher, Vienna, Austria); reagents—Cytofix–Cytoperm permeabilization Kit (Thermofisher, Vienna, Austria), FITC active caspase-3 apoptosis kit (BD Biosciences Kit; Allschwil, Germany), and WST-1 cell proliferation reagent (Abcam; Cambridge, UK); chemicals—alpha-ketoglutarate (Sigma, Vienna, Austria), 5-hydroxymethyl-furfurale (5-HMF, Evonik Operation, Darmstadt, Germany), guanidine-HCl, butyl-hydroxy-toluene (BHT; Sigma Aldrich; Vienna, Austria), and di-nitro-phenyl-hydrazine (DNPH) (Altmann Analytics, Munich, Germany); flasks (Falco® Cell Culture; Corning Incorporated, 14831 New York, NY, USA).
Greilberger J., Erlbacher K., Stiegler P., Wintersteiger R, & Herwig R. (2023). Different RONS Generation in MTC-SK and NSCL Cells Lead to Varying Antitumoral Effects of Alpha-Ketoglutarate + 5-HMF. Current Issues in Molecular Biology, 45(8), 6503-6525.
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3

Rescue Metabolites Enhance Cell Survival

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Cells were seeded in 384-well plates at concentration of 1000–2000 cells/well and allowed to adhere overnight. The next morning growth medium was replaced by fresh growth medium (RPMI-1640+ 10% HI-FBS) containing the following rescue intermediates: nicotinamide mononucleotide (NMN, 25 μM), nicotinic acid (NA, 25 μM), adenine (100 μM, Sigma-Aldrich), uridine (100 μM, Sigma-Aldrich), nucleoside mix (1x, EmbryoMax Nucleosides), dimethyl-alpha-ketoglutarate (50 μM, Sigma-Aldrich), alpha-ketoglutarate (50 μM, Sigma-Aldrich), dimethyl-succinate (50 μM, Sigma-Aldrich), succinate (50 μM, Sigma-Aldrich), dimethyl-fumarate (50 μM, Sigma-Aldrich), fumarate (50 μM, Sigma-Aldrich), dimethyl-malate (50 μM, Sigma-Aldrich), malate (50 μM, Sigma-Aldrich), glutamine (2mM, Gibco) and sodium pyruvate (1mM, Gibco), 3-phosphoglyceric acid (50 μM, 3-PGA, Sigma-Aldrich), N-acetylcysteine (10mM, NAC, Sigma-Aldrich). After replacing the medium with fresh growth medium in presence or absence of rescue metabolites, FK866 and BH3 mimetic drugs were added using the HP D300e Digital Dispenser (Hewlett-Packard Development Company). Cells were treated for 72 h before annexin-V-Hoechst staining was performed as described under measurements of cell death.
Daniels V.W., Zoeller J.J., van Gastel N., McQueeney K.E., Parvin S., Potter D.S., Fell G.G., Ferreira V.G., Yilma B., Gupta R., Spetz J., Bhola P.D., Endress J.E., Harris I.S., Carrilho E., Sarosiek K.A., Scadden D.T., Brugge J.S, & Letai A. (2021). Metabolic perturbations sensitize triple-negative breast cancers to apoptosis induced by BH3 mimetics. Science signaling, 14(686), eabc7405.
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4

Metabolic Analysis of Cellular Compounds

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Cordycepin, streptozotocin (STZ), pentobarbital sodium, and TCA cycle standard samples (oxaloacetate, alpha-ketoglutarate, and citrate) were purchased from Sigma (St. Louis, USA).
Parunyakul K., Srisuksai K., Charoenlappanit S., Phaonakrop N., Roytrakul S, & Fungfuang W. (2021). Metabolic impacts of cordycepin on hepatic proteomic expression in streptozotocin-induced type 1 diabetic mice. PLoS ONE, 16(8), e0256140.
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5

Enzyme Purification and Characterization

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Purified enzymes were purchased from Sigma, including LDH from bovine heart (L2625), LDH from rabbit muscle (L2500), and MDH from porcine heart (M1567). Recombinant human enzymes were purchased from Abcam, including LDHA (ab93699), MDH1 (ab99244), MDH2 (ab99238), IDH1 (ab113858), and PHGDH (ab198455). Substrates, cofactors, and inhibitors were purchased from Sigma, including pyruvate, oxaloacetate, alpha-ketoglutarate, dimethyl-alpha-ketoglutarate, L-lactate, D-lactate, L-malate, D-malate, L-2-hydroxyglutarate, D-2-hydroxyglutarate, alpha-ketobutyrate, oxopentanoic acid, oxoadipic acid, ketohexanoic acid, phenylpyruvate, hydroxyphenylpyruvate, NADH, NADPH, and oxamate. FLAG-tagged wildtype and Q100R mutant LDHA constructs were cloned into the PCDNA3.1(+) vector (Addgene) by standard site-directed mutagenesis and verified by Sanger sequencing.
Intlekofer A.M., Wang B., Liu H., Shah H., Carmona-Fontaine C., Rustenburg A.S., Salah S., Gunner M.R., Chodera J.D., Cross J.R, & Thompson C.B. (2017). L-2-hydroxyglutarate production arises from non-canonical enzyme function at acidic pH. Nature chemical biology, 13(5), 494-500.
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6

Isotope Standards for Analytical Methods

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Authentic standards of d7-glucose, d3-leucine, d8-phenylalanine and d5-tryptophan were purchased from Cambridge Isotope Laboratories (Andover, MA). D5-hippuric acid, d5-indole acetic acid and d9-progesterone were procured from C/D/N Isotopes, Inc. (Pointe-Claire, Quebec). Bromophenylalanine was provided by Sigma-Aldrich Co. LLC. (St. Louis, MO) and amitriptyline was from MP Biomedicals, LLC. (Aurora, OH). Recovery standards of DL-2-fluorophenylglycine and DL-4-chlorophenylalanine were from Aldrich Chemical Co. (Milwaukee, WI). Tridecanoic acid was purchased from Sigma-Aldrich (St. Louis, MO) and d6-cholesterol was from Cambridge Isotope Laboratories (Andover, MA). Standards for the HILIC dilution series of alpha-ketoglutarate, ATP, malic acid, NADH and oxaloacetic acid were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO) while succinic acid, pyruvic acid and NAD+ were purchased from MP Biomedicals, LLC. (Santa Ana, CA).
Al-Khelaifi F., Diboun I., Donati F., Botrè F., Alsayrafi M., Georgakopoulos C., Yousri N.A., Suhre K, & Elrayess M.A. (2018). Metabolomics profiling of xenobiotics in elite athletes: relevance to supplement consumption. Journal of the International Society of Sports Nutrition, 15, 48.
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7

Cell Proliferation and Apoptosis Assay

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Media: RPMI 1640, FBS, DMEM, antibiotic–antimycotic solution (Thermofisher, Vienna, Austria); reagents: Cytofix–Cytoperm permeabilization Kit (Thermofisher, Vienna, Austria), FITC Active Caspase-3 Apoptosis Kit (BD Biosciences Kit; Allschwil, Germany), and WST-1 Cell Proliferation Reagent (Abcam; Cambridge, UK); chemicals: alpha-ketoglutarate, hydrochloric acid, mangan oxide and sodium nitrite (Sigma Aldrich, Vienna, Austria), and 5-hydroxymethyl-furfurale (5-HMF, Evonik Operation, Darmstadt, Germany); flasks (Falco® Cell Culture; Corning Incorporated, New York, NY 14831, USA) were used.
Greilberger J., Herwig R., Kacar M., Brajshori N., Feigl G., Stiegler P, & Wintersteiger R. (2022). Alpha-Ketoglutarate or 5-HMF: Single Compounds Effectively Eliminate Leukemia Cells via Caspase-3 Apoptosis and Antioxidative Pathways. International Journal of Molecular Sciences, 23(16), 9034.
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8

Thermal Stability Profiling of RsbU

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RsbU45–313 was purified as described above and buffer exchanged into PBS (Corning). DSF were performed with SyproOrange (Invitrogen) in 384-well plate (Roche) format [46 (link)]. The following potential ligands were tested: succinate, malonate, glutamate, alpha-ketoglutarate, fumarate, oxaloacetate, malate, 2-phosphoglycerate, glucose, pyruvate, phosphoenolpyruvate, and ATP (Sigma-Aldrich). All ligands were dissolved in PBS. Compounds were added to each well, followed by DSF buffer HEPES-NaOH pH7.5 (100mM), and a 10X SyproOrange dye. Reliable baselines for Tm shifts were established using 10X SyproOrange and 10 μM RsbU45–313. The mixture was heated from 20 to 85 °C. Melting curves were analyzed on Roche Tm Analysis Software.
Soules K.R., Dmitriev A., LaBrie S.D., Dimond Z.E., May B.H., Johnson D.K., Zhang Y., Battaile K.P., Lovell S, & Hefty P.S. (2019). Structural and ligand binding analyses of the periplasmic sensor domain of RsbU in Chlamydia trachomatis supports role in TCA cycle regulation. Molecular microbiology, 113(1), 68-88.
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9

RsbU Protein Interaction Analysis

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RsbU45–313 (30 μM) was purified as described above and buffer exchanged into PBS (Corning). Alpha-ketoglutarate, malate, oxaloacetate, malonate, and succinate (Sigma-Aldrich) were dissolved in the same PBS used for the buffer exchange of the RsbU45–313 protein at a concentration of 30 mM. ITC was performed on a MicroCal PEAQ-ITC (Malvern Panalytical and analyzed using MicroCal ITC Analysis Software (version 1.21).
Soules K.R., Dmitriev A., LaBrie S.D., Dimond Z.E., May B.H., Johnson D.K., Zhang Y., Battaile K.P., Lovell S, & Hefty P.S. (2019). Structural and ligand binding analyses of the periplasmic sensor domain of RsbU in Chlamydia trachomatis supports role in TCA cycle regulation. Molecular microbiology, 113(1), 68-88.
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10

Enzyme Purification and Characterization

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Purified enzymes were purchased from Sigma, including LDH from bovine heart (L2625), LDH from rabbit muscle (L2500), and MDH from porcine heart (M1567). Recombinant human enzymes were purchased from Abcam, including LDHA (ab93699), MDH1 (ab99244), MDH2 (ab99238), IDH1 (ab113858), and PHGDH (ab198455). Substrates, cofactors, and inhibitors were purchased from Sigma, including pyruvate, oxaloacetate, alpha-ketoglutarate, dimethyl-alpha-ketoglutarate, L-lactate, D-lactate, L-malate, D-malate, L-2-hydroxyglutarate, D-2-hydroxyglutarate, alpha-ketobutyrate, oxopentanoic acid, oxoadipic acid, ketohexanoic acid, phenylpyruvate, hydroxyphenylpyruvate, NADH, NADPH, and oxamate. FLAG-tagged wildtype and Q100R mutant LDHA constructs were cloned into the PCDNA3.1(+) vector (Addgene) by standard site-directed mutagenesis and verified by Sanger sequencing.
Intlekofer A.M., Wang B., Liu H., Shah H., Carmona-Fontaine C., Rustenburg A.S., Salah S., Gunner M.R., Chodera J.D., Cross J.R, & Thompson C.B. (2017). L-2-hydroxyglutarate production arises from non-canonical enzyme function at acidic pH. Nature chemical biology, 13(5), 494-500.
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